SELECTED PUBLICATIONS

Latent cytokines for targeted therapy of inflammatory disorders.

Mullen L, Adams G, Layward L, Vessillier S, Annenkov A, Mittal G, Rigby A, Sclanders M, Baker D, Gould D, Chernajovsky Y. Expert Opin Drug Deliv. 2014 Jan;11(1):101-10. doi: 10.1517/17425247.2014.863872. Epub 2013 Dec 3. Review. PMID: 24294995 [PubMed - indexed for MEDLINE]

ABSTRACT

INTRODUCTION:
The use of cytokines as therapeutic agents is important, given their potent biological effects. However, this very potency, coupled with the pleiotropic nature and short half-life of these molecules, has limited their therapeutic use. Strategies to increase the half-life and to decrease toxicity are necessary to allow effective treatment with these molecules.
AREAS COVERED:
A number of strategies are used to overcome the natural limitations of cytokines, including PEGylation, encapsulation in liposomes, fusion to targeting peptides or antibodies and latent cytokines. Latent cytokines are engineered using the latency-associated peptide of transforming growth factor-β to produce therapeutic cytokines/peptides that are released only at the site of disease by cleavage with disease-induced matrix metalloproteinases. The principles underlying the latent cytokine technology are described and are compared to other methods of cytokine delivery. The potential of this technology for developing novel therapeutic strategies for the treatment of diseases with an inflammatory-mediated component is discussed.
EXPERT OPINION:
Methods of therapeutic cytokine delivery are addressed. The latent cytokine technology holds significant advantages over other methods of drug delivery by providing simultaneously increased half-life and localised drug delivery without systemic effects. Cytokines that failed clinical trials should be reassessed using this delivery system.

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Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-β.

Mullen L, Rigby A, Sclanders M, Adams G, Mittal G, Colston J, Fatah R, Subang C, Foster J, Francis-West P, Köster M, Hauser H, Layward L, Vessillier S, Annenkov A, Al-Izki S, Pryce G, Bolton C, Baker D, Gould DJ, Chernajovsky Y. Expert Opin Drug Deliv. 2014 Jan;11(1):5-16. doi: 10.1517/17425247.2013.839655. Epub 2013 Sep 27. PMID: 24073618 [PubMed - indexed for MEDLINE]

ABSTRACT

OBJECTIVES:
Targeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-β with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes.
METHODS:
Mature cytokines cloned downstream of LAP and a MMP cleavage site were expressed in 293T cells and assessed for latency and biological activity by Western blotting and bioassay.
RESULTS:
We demonstrate here that fusion proteins of TGF-β, erythropoietin, IL-1ra, IL-10, IL-4, BMP-7, IGF1 and IL-17 were rendered latent by fusion to LAP, requiring cleavage to become active in respective bioassays. As further proof of principle, we also show that delivery of engineered TGF-β can inhibit experimental autoimmune encephalomyelitis and that this approach can be used to efficiently deliver cytokines to the brain and spinal cord in mice with this disease.
CONCLUSIONS:
The latent cytokine approach can be successfully applied to a range of molecules, including cytokines of different molecular structure and mass, growth factors and a cytokine antagonist.

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Generation of an efficiently secreted, cell penetrating NF-κB inhibitor.

Koutsokeras A, Purkayastha N, Rigby A, Subang MC, Sclanders M, Vessillier S, Mullen L, Chernajovsky Y, Gould D. FASEB J. 2014 Jan;28(1):373-81. doi: 10.1096/fj.13-236570. Epub 2013 Sep 26. Erratum in: FASEB J. 2014 Jun;28(6):2737. Purkayashta, Nirupam [corrected to Purkayastha, Nirupam]. PMID: 24072781 [PubMed - indexed for MEDLINE] Free Article

ABSTRACT

Gene therapy is a powerful approach to treat disease locally. However, if the therapeutic target is intracellular, the therapeutic will be effective only in the cells where the therapeutic gene is delivered. We have engineered a fusion protein containing an intracellular inhibitor of the transcription factor NF-κB pathway that can be effectively secreted from producing cells. This fusion protein is cleaved extracellularly by metalloproteinases allowing release of a protein transduction domain (PTD) linked to the NF-κB inhibitor for translocation into neighboring cells. We show that engineered molecules can be efficiently secreted (>80%); are cleaved with matrix metalloprotease-1; inhibit NF-κB driven transcription in a biological assay with a human reporter cell line; and display significant inhibition in mouse paw inflammation models when delivered by lentivirus or secreting cells. No inhibition of NF-κB transcription or therapeutic effect was seen using molecules devoid of the PTD and NF-κB inhibitory domains. By creating a fusion protein with an endogenous secretion partner, we demonstrate a novel approach to efficiently secrete PTD-containing protein domains, overcoming previous limitations, and allowing for potent paracrine effects.

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A comparative study of matrix metalloproteinase and aggrecanase mediated release of latent cytokines at arthritic joints.

Mullen L, Adams G, Foster J, Vessillier S, Köster M, Hauser H, Layward L, Gould D, Chernajovsky Y. Ann Rheum Dis. 2014 Sep;73(9):1728-36. doi: 10.1136/annrheumdis-2013-203513. Epub 2013 Jun 27. PMID: 23813971 [PubMed - indexed for MEDLINE]

ABSTRACT

BACKGROUND:
Latent cytokines are engineered by fusing the latency associated peptide (LAP) derived from transforming growth factor-β (TGF-β) with the therapeutic cytokine, in this case interferon-β (IFN-β), via an inflammation-specific matrix metalloproteinase (MMP) cleavage site.
OBJECTIVES:
To demonstrate latency and specific delivery in vivo and to compare therapeutic efficacy of aggrecanase-mediated release of latent IFN-β in arthritic joints to the original MMP-specific release.
METHODS:
Recombinant fusion proteins with MMP, aggrecanase or devoid of cleavage site were expressed in CHO cells, purified and characterised in vitro by Western blotting and anti-viral protection assays. Therapeutic efficacy and half-life were assessed in vivo using the mouse collagen-induced arthritis model (CIA) of rheumatoid arthritis and a model of acute paw inflammation, respectively. Transgenic mice with an IFN-regulated luciferase gene were used to assess latency in vivo and targeted delivery to sites of disease.
RESULTS:
Efficient localised delivery of IFN-β to inflamed paws, with low levels of systemic delivery, was demonstrated in transgenic mice using latent IFN-β. Engineering of latent IFN-β with an aggrecanase-sensitive cleavage site resulted in efficient cleavage by ADAMTS-4, ADAMTS-5 and synovial fluid from arthritic patients, with an extended half-life similar to the MMP-specific molecule and greater therapeutic efficacy in the CIA model.
CONCLUSIONS:
Latent cytokines require cleavage in vivo for therapeutic efficacy, and they are delivered in a dose dependent fashion only to arthritic joints. The aggrecanase-specific cleavage site is a viable alternative to the MMP cleavage site for the targeting of latent cytokines to arthritic joints. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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Increased disulphide dimer formation of latent associated peptide fusions of TGF-β by addition of L-cystine.

Mullen LM, Adams G, Chernajovsky Y. J Biotechnol. 2012 Oct 31;161(3):269-77. doi: 10.1016/j.jbiotec.2012.07.003. Epub 2012 Jul 23. PMID: 22835852 [PubMed - indexed for MEDLINE]

Abstract

The development of novel protein therapeutics relies on the ability to express appreciable amounts of correctly folded recombinant proteins. Latent IFN-β is engineered using the latency-associated peptide (LAP) of transforming growth factor β1 (TGF-β1) to maintain IFN-β in a biologically inactive form until such time as it is released at sites of inflammation by matrix metalloproteinase activity (see Adams et al., 2003). CHO cells cultured in suspension were used for expression of latent IFN-β to allow medium scale transient transfection. However, the recombinant protein expressed in this system consisted of a mixture of properly linked disulphide dimers and monomers. The ratio of dimer:monomer produced could be significantly altered towards increased dimer production by the addition of L-cystine to the CHO culture medium. The total yield of latent IFN-β was increased by co-transfection of plasmid coding for the simian virus (SV) 40 large T antigen to the plasmid with the SV40 origin of replication expressing latent IFN-β DNA. These results provide valuable new insights for developing protocols to produce substantial quantities of latent cytokine dimers in CHO cells in suspension.

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Molecular engineering of short half-life small peptides (VIP, αMSH and γ₃MSH) fused to latency-associated peptide results in improved anti-inflammatory therapeutics.

Vessillier S, Adams G, Montero-Melendez T, Jones R, Seed M, Perretti M, Chernajovsky Y. Ann Rheum Dis. 2012 Jan;71(1):143-9. doi: 10.1136/annrheumdis-2011-200100. Epub 2011 Oct 13. PMID: 21998117 [PubMed - indexed for MEDLINE]

Abstract

OBJECTIVE:
To facilitate the targeting to inflammation sites of small anti-inflammatory peptides, with short half-lives, by fusion with the latency-associated peptide (LAP) of transforming growth factor β1 through a cleavable matrix metalloproteinase (MMP) linker. This design improves efficacy, overcoming the limitations to their clinical use.
METHODS:
We generated latent forms of vasoactive intestinal peptide (VIP), α-melanocyte-stimulating hormone (MSH) and γ(3)MSH by fusion to LAP through an MMP cleavage site using recombinant DNA technology. The biological activities of these latent therapeutics were studied in vivo using monosodium urate (MSU)-induced peritonitis and collagen-induced arthritis (CIA) models. We assessed gene therapy and purified protein therapy.
RESULTS:
The recruitment of the polymorphonuclear cells induced by MSU injection into mouse peritoneal cavity was reduced by 35% with γ(3)MSH (1 nmol), whereas administration of a much lower dose of purified latent LAP-MMP-γ(3)MSH (0.03 nmol) attenuated leucocyte influx by 50%. Intramuscular gene delivery of plasmids coding LAP-MMP-VIP and LAP-MMP-αMSH at disease onset reduced the development of CIA compared with LAP-MMP, which does not contain any therapeutic moiety. Histological analysis confirmed a significantly lower degree of inflammation, bone and cartilage erosion in groups treated with LAP-MMP-VIP or LAP-MMP-αMSH. Antibody titres to collagen type II and inflammatory cytokine production were also reduced in these two groups.
CONCLUSION:
Incorporation of small anti-inflammatory peptides within the LAP shell and delivered as recombinant protein or through gene therapy can control inflammatory and arthritic disease. This platform delivery can be developed to control human arthritides and other autoimmune diseases.

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Latent cytokines: development of novel cleavage sites and kinetic analysis of their differential sensitivity to MMP-1 and MMP-3.

Vessillier S, Adams G, Chernajovsky Y. Protein Eng Des Sel. 2004 Dec;17(12):829-35. Epub 2005 Feb 11. PMID: 15708865 [PubMed - indexed for MEDLINE]

Abstract

We have engineered a latent mouse interferon beta (mIFNbeta) using the latency associated peptide (LAP) of transforming growth factor beta1 (TGF-beta1) to protect the cytokine and avoid its interaction with its receptors. This approach improves the pharmacokinetic properties and reduces the pleiotropic effects limiting the current therapeutic use of cytokines. IFNbeta was fused to the LAP using two flexible linkers flanking a matrix metalloproteinase (MMP) cleavage site for the specific release of IFNbeta at disease sites. In order to improve the hydrolysis rate of the cleavage site, 15 different cleavable linkers were introduced in the LAP-mIFNbeta construct. The kinetic parameters relative to the linker cleavage by MMP-1 and MMP-3 were measured by an ELISA method. Among the modifications done, one of the constructs bearing the activation site of pro-MMPs was the best substrate for both enzymes. The introduction of a hydrophilic sequence derived from the furin cleavage site of the anthrax toxin protective antigen increased the sensitivity to MMP-3 to up to 29-fold. These data suggest that this strategy could be useful for improving the effectiveness of the delivery and targeting of protein therapeutics.

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Targeting therapeutic molecules to sites of disease.

Chernajovsky Y, Vessillier S, Adams G. Discov Med. 2004 Feb;4(20):30-2. PMID: 20705016 [PubMed]

ABSTRACT

Extract: The holy grail of drug development is to design a magic bullet that will deliver a therapeutic agent only to the site of disease with minimal side effects. This is of great importance in particular when it refers to biological agents such as cytokines that have pleiotropic actions in different tissues. Cytokines are local mediators of cell-to-cell communication. Their expression is transient and they have a short half-life. Therefore, to overcome the pharmacokinetic limitations of cytokines, they are given subcutaneously to reduce side effects. However, by this route they lose their potency. In order to use cytokines for therapeutic purposes they have to be administered frequently at high doses to achieve biologically active concentrations locally at the required sites of a disease.

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Targeting cytokines to inflammation sites.

Adams G, Vessillier S, Dreja H, Chernajovsky Y. Nat Biotechnol. 2003 Nov;21(11):1314-20. Epub 2003 Oct 5. PMID: 14528315 [PubMed - indexed for MEDLINE]

ABSTRACT

To increase the half-life of a cytokine and target its activation specifically to disease sites, we have engineered a latent cytokine using the latency-associated protein (LAP) of transforming growth factor-beta 1 (TGF-beta 1) fused via a matrix metalloproteinase (MMP) cleavage site to interferon (IFN)-beta at either its N or C terminus. The configuration LAP-MMP-IFN-beta resembles native TGF-beta and lacks biological activity until cleaved by MMPs, whereas the configuration IFN-beta-MMP-LAP is active. LAP provides for a disulfide-linked shell hindering interaction of the cytokine with its cellular receptors, conferring a very long half-life of 55 h in vivo. Mutations of the disulfide bonds in LAP abolish this latency. Samples of cerebrospinal fluid (CSF) or synovial fluid from patients with inflammatory diseases specifically activate the latent cytokine, whereas serum samples do not. Intramuscular injection in arthritic mice of plasmid DNA encoding these constructs demonstrated a greater therapeutic effect of the latent as compared to the active forms.

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